Interrelationships between protein kinases and spectrin phosphorylation in human erythrocytes.

Casein kinase and histone kinase(s) are solubilized from human erythrocyte membranes by buffered ionic solutions (0.1 mM EDTA and subsequent 0.8 M NaCl, pH 8) containing 0.2% Triton X-100. Casein kinase is separated from histone kinase(s) by submitting the crude extracts directly to chromatography on a phosphocellulose column, eluted with a continuous linear gradient of potassium phosphate buffer, pH 7.0, containing 0.2% Triton X-100. Under these conditions, the membrane-bound casein kinase activity is almost completely recovered into a quite stable preparation, free of histone kinase activity. In contrast, it undergoes a dramatic loss of activity when the extraction and the subsequent phosphocellulose chromatography are carried out with buffers which do not contain Triton X-100. Isolated spectrin, the most abundant membrane protein, is phosphorylated, in the presence of [gamma-32P]ATP, only by casein kinase while histone kinase is ineffective. Only the smaller subunit (band II) of isolated spectrin (and not the larger one (band I) is involved in such a phosphorylation process, as in the endogenous phosphorylation occurring in intact erythrocytes.

Multiple forms of cytosol and membrane-bound protein kinase activity in human erythrocytes.

Both cytosol and membranes of human erythrocytes display protein kinase activity towards exogenous protein substrates such as casein, phosvitin and histones. The histone kinase activity, unlike casein kinase, of both cytosol and membranes is increased by cyclic AMP. The protein kinase forms removed from the membranes with 0.7 M NaCl, phosphorylate only serine residues of both casein and histones through a mechanism cyclic AMP-independent. The protein kinase activity located in the cytosol (hemolysate) is due also to enzyme forms phosphorylating both serine and threonine residues of casein, in addition to forms phosphorylating only serine residues of casein and histones. Also the cytosol kinase forms, once partially purified by Sepharose 6B filtration, appear to be cyclic AMP-independent.

Maturation-related change of the protein kinase activity in the rabbit red blood cells.

The protein kinase activity of rabbit reticulocyte and erythrocyte hemolysate is due to multiple forms (casein kinases and histone kinases) which have been partially purified by Sepharose 6B filtration followed by phosphocellulose chromatography in the presence of 0.2% Triton X-100. The casein kinase activity is resolved by such a procedure into two forms differing in catalytic properties: i.e. the casein kinase TS phosphorylates both serine and threonine residues of casein, while the casein kinase S phosphorylates only serine residues. The comparative results indicate that during differentiation and maturation of rabbit red blood cells multiple cytosol protein kinase activities markedly decrease, while the casein kinase S does not change significantly.

Partial characterization of cytosol and membrane-bound protein kinases in hereditary spherocytosis erythrocytes.

The protein kinase activity located in the cytosol of hereditary spherocytosis erythrocytes is due to multiple forms which can be resolved by Sepharose 6B filtration at high ionic strength into two fractions phosphorylating the whole casein on different sites. The membrane-bound protein kinases, solubilized by 0.7 M NaCl, display an elution volume from Sepharose column and a phosphorylation behaviour towards casein quite similar to those of the more retarded fraction of hemolysate. When compared with the multiple protein kinase forms from normal human erythrocytes, no significant difference has been found.

Endogenous phosphorylation of membrane proteins in normal and in hereditary spherocytosis erythrocytes.

The phosphorylation state of the proteins in hereditary spherocytosis erythrocyte membranes, incubated in the presence of [gamma-32P]ATP, appears to be different from that in normal ones. This is indicated by the finding that in the two types of erythrocyte membranes the ratios between the 32P-labeling of their phosphorylserine and phosphorylthreonine residues were different.

Characteristics and behaviour of arylesterase in human serum and liver.

Human arylesterase is localized in liver microsomes where the presence of different electrophoretic bands corresponding to the serum bands can be recognized. Serum arylesterase is mainly a result of liver activity and its high level might be explained by a low rate of elimination in urine. The behaviour of arylesterase towards inhibitors shows certain similarities to that of some of the proteases, such as trypsin. The clinical value of serum arylesterase determination in assessing liver function is confirmed by its isoenzyme behaviour in cirrhosis and porto-caval shunt.